Not Applicable
The present invention relates to immunomodulatory compositions comprising an immunostimulatory oligonucleotide sequence (ISS) in which at least one base has been substituted with a base modified by the addition to C-5 and/or C-6 on cytosine with an electron-withdrawing moiety. It also relates to the administration of said ISS to modulate an immune response.
The type of immune response generated to infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The Th1 subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subset functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally determined by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by Th1 and Th2 cells are believed to reflect different biological functions of these two subsets.
The Th1 subset may be particularly suited to respond to viral infections and intracellular pathogens because it secretes IL-2 and IFN-xcex3, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. In general, Th1 and Th2 cells secrete distinct patterns of cytokines and so one type of response can moderate the activity of the other type of response. A shift in the Th1/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.
Immunization of a host animal against a particular antigen has been accomplished traditionally by repeatedly vaccinating the host with an immunogenic form of the antigen. While most current vaccines elicit effective humoral (antibody, or xe2x80x9cTh2-typexe2x80x9d) responses, they fail to elicit cellular responses (in particular, major histocompatibility complex (MHC) class I-restricted CTL, or xe2x80x9cTh1 -typexe2x80x9d responses) which are generally absent or weak. For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Moreover, antibody responses are inappropriate in certain indications, most notably in allergy where an antibody response can result in anaphylactic shock. Proposed vaccines using small peptides derived from the target antigen and other currently used antigenic agents that avoid use of potentially infective intact viral particles, do not always elicit the immune response necessary to achieve a therapeutic effect. The lack of a therapeutically effective human immunodeficiency virus (HIV) vaccine is an unfortunate example of this failure.
Protein-based vaccines typically induce Th2-type immune responses, characterized by high titers of neutralizing antibodies but without significant cell-mediated immunity. In contrast, intradermal delivery of xe2x80x9cnakedxe2x80x9d, or uncomplexed, DNA encoding an antigen stimulates immune responses to the antigen with a Th1-type bias, characterized by the expansion of CD4+ T cells producing IFN-xcex3 and cytotoxic CD8+ T cells. Manickan et al. (1995) J. Immunol. 155:250-265; Xiang et al. (1995) Immunity 2:129-135; Raz et al. (1995) Proc. Natl. Acad. Sci. USA 93:5141-5145; and Briode et al. (1997) J. Allergy Clin. Immunol. 99:s129. Injection of antigen-encoding naked DNA reproducibly induces both humoral and cellular immune responses against the encoded antigens. Pardoll and Beckerleg (1995) Immunity 3:165-169. DNA vaccines can provide a new approach to infectious disease prophylaxis. See, for instance, Dixon (1995) Bio/Technology 13:420 and references cited therein.
Certain types of DNA, without being translated, have been shown to stimulate immune responses. Bacterial DNA induces anti-DNA antibodies in injected mice, as well as cytokine production by macrophage and natural killer (NK) cells. Pisetsky (1996) J. Immunol. 156:421-423; Shimada et al. (1986) Jpn. J. Cancer Res. 77:808-816; Yamamoto et al. (1992a) Microbiol. Immunol. 36:983-897; and Cowdery et al. (1996) J. Immunol. 156:4570-4575.
B cell and NK cell activation properties of bacterial DNA have been associated with short (6 base pair hexamer) sequences that include a central unmethylated CpG dinucleotide. Yamamoto et al. (1992a); and Krieg et al. (1995) Nature 374:546-549. Oligonucleotides comprising a CpG sequence flanked by two 5xe2x80x2 purines and two 3xe2x80x2 pyrimidines have been shown to be most potent in B cell and NK cell stimulation. For example, when a variety of oligonucleotides comprising hexamers were tested for their ability to augment the NK cell activity of mouse spleen cells, the most immunogenic hexamers included AACGTT, AGCGCT, GACGTC. Yamamoto et al. (1992b) J. Immunol. 148:4072-4076. In a study in which B cell activation was measured in response to oligonucleotides, the most stimulatory hexamer sequences (e.g., AACGTC, AACGTT, GACGTC, GACGTT) also matched the sequence of 5xe2x80x2-purine, purine, CG, pyrimidine, pyrimidine-3xe2x80x2. Krieg et al. (1995).
Bacterial DNA stimulated macrophages to produce IL-12 and TNF-xcex1. These macrophage-produced cytokines were found to induce the production of IL-12 and IFN-xcex3 from splenocytes. Halpern et al. (1996) Cell. Immunol. 167:72-78. In vitro treatment of splenocytes with either bacterial DNA or CpG containing oligonucleotides induced the production of IL-6, IL-12 and IFN-xcex3. Klinman et al. (1996) Proc. Natl. Acad. Sci. USA 93:2879-2883. Production of all of these cytokines is indicative of induction of a Th1-type immune response rather than a Th2-type response.
Todate, no clear consensus has been reached on the sequences both necessary and sufficient of immune stimulation. A recent study which examined induction of NK activity in response to CpG containing-oligonucleotides suggested that the unmethylated CpG motif was necessary but not sufficient for oligonucleotide induction of NK lytic activity. Ballas et al. (1996) J. Immunol. 157:1840-1845. Sequences flanking the CpG appeared to influence the immunostimulatory activity of an oligonucleotide. Immunostimulatory activity of immunostimulatory sequences appears to be independent of adenosine-methylation, and whether the nucleotide is single or double-stranded. See, for example, Tokunaga et al. (1989) Microbiol Immunol. 33:929; Tokunaga et al. (1992) Microbiol. Immunol. 36:55-66; Yamamoto et al. (1992b); Messina et al. (1993) Cell. Immunol. 147:148-157; and Sato et al. (1996) Science 273:352-354. Oligonucleotide length also does not seem to be a factor, as double-stranded DNA 4 kb long (Sato et al. (1996)) or single-stranded DNA as short as 15 nucleotides in length (Ballas et al. (1996)) illicited immune responses; though if oligonucleotide length was reduced below 8 bases or if the DNA was methylated with CpG methylase, immunostimulatory activity was abolished. Krieg et al. (1995).
Allergic responses, including those of allergic asthma, are characterized by an early phase response, which occurs within seconds to minutes of allergen exposure and is characterized by cellular degranulation, and a late phase response, which occurs 4 to 24 hours later and is characterized by infiltration of eosinophils into the site of allergen exposure. Specifically, during the early phase of the allergic response, activation of Th2-type lymphocytes stimulates the production of antigen-specific IgE antibodies, which in turn triggers the release of histamine and other mediators of inflammation from mast cells and basophils. During the late phase response, IL-4 and IL-5 production by CD4+ Th2 cells is elevated. These cytokines appear to play a significant role in recruiting eosinophils into site of allergen exposure, where tissue damage and dysfunction result.
Antigen immunotherapy for allergic disorders involves the subcutaneous injection of small, but gradually increasing amounts, of antigen. Such immunization treatments present the risk of inducing IgE-mediated anaphylaxis and do not address the cytokine-mediated events of the allergic late phase response.
Vaccination with certain DNA containing immunostimulatory motifs induces an immune response with a Th1-type bias. For example, mice injected intradermally with Escherichia coli (E. coli) xcex2-galactosidase (xcex2-Gal) in saline or in the adjuvant alum responded by producing specific IgG1 and IgE antibodies, and CD4+ cells that secreted IL-4 and IL-5, but not IFN-xcex3, demonstrating that the T cells were predominantly of the Th2 subset. However, mice injected intradermally (or with a tyne skin scratch applicator) with plasmid DNA (in saline) encoding xcex2-Gal and containing an ISS responded by producing IgG2a antibodies and CD4+ cells that secreted IFN-xcex3, but not IL-4 and IL-5, demonstrating that the T cells were predominantly of the Th1 subset. Moreover, specific IgE production by the plasmid DNA-injected mice was reduced 66-75%. Raz et al. (1996) Proc. Natl. Acad. Sci. USA 93:5141-5145. In general, the response to naked DNA immunization is characterized by production of IL-2, TNFxcex1 and IFN-xcex3 by antigen-stimulated CD4+ T cells, which is indicative of a Th1-type response. This is particularly important in treatment of allergy and asthma as shown by the decreased IgE production.
In another example, the presence of an immunostimulatory sequence, such as the palindromic hexamer AACGTT, in an antigen-encoding plasmid vector injected intradermally prompted the production of large amounts of IFN-xcex1, IFN-xcex2 and IL-12. Sato et al. (1996). IFN-xcex1 plays a role in the differentiation of naive T cells toward a Th1-type phenotype, antagonizes Th2 cells, inhibits IgE synthesis, promotes IgG2a production and induces a Th1 phenotype of antigen-specific T cell clones. IL-12 promotes IFN-xcex3 production by T cells and favors maturation of Th1 cells.
It would be useful in treatment of a wide variety of indications to be able to specifically enhance the Th1-type response to a particular antigen while down-regulating the Th2-type response to the same antigen. Treatment or palliation of these indications includes, but is not limited to, tumor therapy, treatment of allergic disorders and induction of a vigorous cellular immune response. The present invention provides compositions comprising oligonucleotide sequences that can be employed in these contexts.
All of the cited literature included in the preceding section, as well as the cited literature included in the following disclosure, are hereby incorporated by reference.
In one embodiment, the ISS comprises a hexameric sequence or hexanucleotide comprising a central CG sequence, where the C residue is modified by the addition to C-5 and/or C-6 with an electron-withdrawing moiety. Preferably, the electron-withdrawing group is a halogen or halogen-containing ligand. Suitable halogens include chlorine, bromine and fluorine. Suitable halogen-containing ligands include, but are not limited to, 5-bromocytosine, 5-fluorocytosine, 5-chlorocytosine, and 5-trifluoromethyl cytosine.
In another embodiment, the modified ISS comprises the general sequence 5xe2x80x2-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine-3xe2x80x2. More preferably, the modified ISS comprises the general sequences selected from the group consisting of AACGTC, AACGTT, AGCGTC, AGCGCT, AGCGTT, GACGTC, GACGTT, and GGCGTT. The modified ISS can also comprise any other physiologically acceptable modification.
In another embodiment, the modified ISS comprises the general sequence 5xe2x80x2-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Cytosine-3xe2x80x2. More preferably, the modified ISS comprises a sequence selected form the group consisting of AACGTTCC and GACGTTCC.
In another embodiment, the modified ISS comprises the general sequence 5xe2x80x2-Purine, Purine, Cytosine, Guanine, Pyrimidine, Pyrimidine, Cytosine, Guanine-3xe2x80x2. More preferably, the modified ISS comprises a sequence selected form the group consisting of AACGTTCG and GACGTTCG.
In another embodiment, the modified ISS comprises the sequence of SEQ ID NO:2.
In another embodiment, the modified ISS comprises the sequence of SEQ ID NO:6.
In another embodiment, the modified ISS comprises the sequence of SEQ ID NO:7.
In another embodiment, the invention provides an immunomodulatory composition comprising a modified ISS and further comprising an antigen.
In another embodiment, the invention provides an immunomodulatory composition comprising a modified ISS in conjunction with a member of the group of immunomodulation facilitators consisting of co-stimulatory molecules, cytokines, chemokines, targeting protein ligand, a trans-activating factor, a peptide, or a peptide comprising a modified amino acid.
In another embodiment, the invention provides an immunomodulatory composition comprising a modified ISS, an antigen and an adjuvant.
The present invention also provides for a method of modulating an immune response comprising the administration of an amount of a modified ISS effective to induce an immune response. Preferably, modulation of an immune response comprises induction of a Th1-type immune response.
In another embodiment, the invention provides methods of treating an individual in need of immune modulation comprising administration of a composition comprising a modified ISS.